Activation of calpain precedes morphological alterations during hydrogen peroxide-induced apoptosis in neuronally differentiated mouse embryonal carcinoma P19 cell line

Ceramides, which are membrane sphingolipids and key mediators of cell-stress responses, are generated by a family of (dihydro) ceramide synthases (Lass1-6/CerS1-6). Here, we report that brain development features significant increases in sphingomyelin, sphingosine, and most ceramide species. In contrast, C 16:0 -ceramide was gradually reduced and CerS6 was down-regulated in mitochondria, thereby implicating CerS6 as a primary ceramide synthase generating C 16:0 -ceramide. Investigations into the role of CerS6 in mitochondria revealed that ceramide synthase down-regulation is associated with dramatically decreased mitochondrial Ca 2؉ -loading capacity, which could be rescued by addition of ceramide. Selective CerS6 complexing with the inner membrane component of the mitochondrial permeability transition pore was detected by immunoprecipitation. This suggests that CerS6-generated ceramide could prevent mitochondrial permeability transition pore opening, leading to increased Ca 2؉ accumulation in the mitochondrial matrix. We examined the effect of high CerS6 expression on cell survival in primary oligodendrocyte (OL) precursor cells, which undergo apoptotic cell death during early postnatal brain development. Exposure of OLs to glutamate resulted in apoptosis that was prevented by inhibitors of de novo ceramide biosynthesis, myriocin and fumonisin B1. Knockdown of CerS6 with siRNA reduced glutamate-triggered OL apoptosis, whereas knockdown of CerS5 had no effect: the pro-apoptotic role of CerS6 was not stimulus-specific. Knockdown of CerS6 with siRNA improved cell survival in response to nerve growth factor-induced OL apoptosis. Also, blocking mitochondrial Ca 2؉ uptake or decreasing Ca 2؉ -dependent protease calpain activity with specific inhibitors prevented OL apoptosis. Finally, knocking down CerS6 decreased calpain activation. Thus, our data suggest a novel role for CerS6 in the regulation of both mitochondrial Ca 2؉ homeostasis and calpain, which appears to be important in OL apoptosis during brain development.

Section: Discussionmentioning

confidence: 99%

Developmentally Regulated Ceramide Synthase 6 Increases Mitochondrial Ca2+ Loading Capacity and Promotes Apoptosis

Novgorodov

Chudakova

Wheeler

et al. 2011

“…Two ubiquitous isozymes are known, -and m-calpains (also called calpains I and calpains II, respectively), which differ in their sensitivity to Ca 2ϩ . Calpains have been reported previously to be activated under oxidative stress conditions in P19, PC12 cells, and in cerebellar granule cells (27)(28)(29). We have previously reported that calcium/calmodulin-dependent protein kinase IV (CaMKIV) plays an anti-apoptotic role in cerebellar granule cells (30).…”

mentioning

confidence: 91%

Amyloid Precursor Protein Family-induced Neuronal Death Is Mediated by Impairment of the Neuroprotective Calcium/Calmodulin Protein Kinase IV-dependent Signaling Pathway

Mbebi¹,

Sée²,

Mercken³

et al. 2002

The aberrant metabolism of ␤-amyloid precursor protein (APP) and the progressive deposition of its derived fragment ␤-amyloid peptide are early and constant pathological hallmarks of Alzheimer's disease. Because APP is able to function as a cell surface receptor, we investigated here whether a disruption of the normal function of APP may contribute to the pathogenic mechanisms in Alzheimer's disease. To this aim, we generated a specific chicken polyclonal antibody directed against the extracellular domain of APP, which is common with the ␤-amyloid precursor-like protein type 2. Exposure of cultured cortical neurons to this antibody (APP-Ab) induced cell death preceded by neurite degeneration, oxidative stress, and nuclear condensation. Interestingly, caspase-3-like protease was not activated in this neurotoxic action suggesting a different mode of cell death than classical apoptosis. Further analysis of the molecular mechanisms revealed a calpain-and calcineurindependent proteolysis of the neuroprotective calcium/ calmodulin-dependent protein kinase IV and its nuclear target protein cAMP responsive element binding protein. These effects were abolished by the G protein inhibitor pertussis toxin, strongly suggesting that APP binding operates via a GTPase-dependent pathway to cause neuronal death. Alzheimer's disease (AD)1 is a devastating neurodegenerative disorder characterized by deposition of ␤-amyloid (A␤) plaques, accumulation of intracellular neurofibrillary tangles, and neuronal cell loss (1). A␤ peptide is generated by proteolysis of the amyloid precursor protein (APP), which is the product of a gene located on human chromosome 21 and on mouse chromosome 16. APP is expressed in most mammalian tissues (2, 3) and is present in at least 10 different spliced variants (4). In the brain, the major isoform generating A␤ is a peptide consisting of 695 residues that contains a single transmembrane domain (5). Although the physiological role of APP is still unclear, several studies have suggested that it is implicated in important physiological functions of neurons, such as neurite outgrowth, synaptogenesis, cell substrate adhesion and neuronal survival (for review see Ref. 6).Up to now, most of the research has been focused on the toxicity of A␤ peptide related to AD. A␤ has been reported to exert a variety of toxic effects on neurons both in vitro (7,8) and in vivo (9). However, it has been reported that mice overproducing A␤1-42 extracellularly showed no neuronal loss (10), thus suggesting that A␤ peptide seems not to be the only constituent in neurotoxicity associated to AD. Previous studies have demonstrated that overexpression of full-length APP induced degeneration of postmitotic neurons derived from embryonal carcinoma cells (11). Moreover, intracellular accumulation of wild-type APP in the rat hippocampus caused a specific type of neuronal degeneration in vivo in the absence of extracellular A␤ deposition (12), and viral vector-mediated overexpression of wild-type APP induced apoptosis-like death of neurons...

“…The increase of calpain activity under oxidative conditions has recently been demonstrated in PC12 cells (11) and in a mouse embryonic carcinoma P19 cell line (12). It thus appears that calpain activation is a general mechanism that operates not only in proliferating and transformed cell lines but also in postmitotic differentiated neurons.…”

Section: H 2 O 2 Induces a Programmed Neuronal Death Distinct Frommentioning

confidence: 94%

“…Their up-regulation by oxidative stress has recently been demonstrated in PC12 pheochromocytoma cells (11) and in the mouse embryonic carcinoma P19 cell line (12). Calpain is one of the most abundant neutral proteinases in the central nervous system and occurs as a proenzyme in association with its endogenous inhibitor, calpastatin.…”

mentioning

confidence: 99%

Oxidative Stress Induces Neuronal Death by Recruiting a Protease and Phosphatase-gated Mechanism

Sée¹,

Loeffler²

2001

Reactive oxygen species (ROS) cause death of cerebellar granule neurons. Here, a 15-min pulse of H 2 O 2 (100 M) induced an active process of neuronal death distinct from apoptosis. Oxidative stress activated a caspaseindependent but calpain-dependent decline of calcium/ calmodulin-dependent protein kinase IV and cAMPresponsive element-binding protein (CREB). Calpain inhibitors restored calcium/calmodulin-dependent protein kinase IV and CREB but did not influence phosphorylated CREB levels or survival, indicating recruitment of an additional dephosphorylation process. Co-treatment with calpain and serine/threonine phosphatase inhibitors restored pCREB levels and rescued neurons. This phosphatase-activated signaling pathway was shown to be dependent on de novo protein synthesis. Further, gene transfer studies revealed that CREB is a common final effector of both apoptosis and ROS-induced death. Our data indicate that dephosphorylation and proteolytic signaling mechanisms underlie ROS-induced programmed cell death.